This document summarizes a study investigating estradiol-conjugated DNA methylating compounds. Three compounds were synthesized and tested for their ability to bind DNA and estrogen receptors, induce DNA methylation, and cause toxicity in breast cancer cells. Compound 2a was found to be the most toxic, producing the highest levels of N3-methyladenine DNA adducts. Fluorescent assays showed compound binding to DNA and varying binding affinities to estrogen receptors. Overall, the results help explain how these compounds can selectively target and damage estrogen receptor-positive breast cancer cells through DNA methylation.

1. DNA binding, DNA Methylation and Cell Toxicity of Estradiol Conjugated DNA
Methylating Compounds
Estrogen Receptor Binding :FRET Assay
Cell Toxicity
DNA Methylation
Design of Molecules
Acknowledgments
Matthew Powella, Sean Millera, Rigel Kishtona, Charles Kellya, Kelly Mastroa, Astrid Linaresb, Manoj Chelvanambib, Vinayak K. Gorec, Giridhar R.
Akkarajub Sridhar Varadarajana,*aDepartment of Chemistry and Biochemistry, University of North Carolina Wilmington, Wilmington, NC, bDepartment of
Biology, Texas Christian University, Fort Worth, TX, cQuality Chemical Laboratories, Wilmington, NC.
Abstract
Estradiol conjugated compounds that can target estrogen receptor positive cells and
produce lethal N3-methyladenine adducts in these cells have been synthesized and
investigated1. Prior studies with these DNA methylating compounds had shown that the
ability of these molecules to produce N3-methyladenine adducts was crucially dependent
on the composition of the linker unit connecting the DNA methylating moiety to the
estradiol unit. The DNA binding properties of this class of molecules was investigated
using stable non-methylating analogs, and the variation in the DNA binding ability of the
compounds was compared to the corresponding DNA methylating ability. Cell toxicity
studies were conducted using MCF-7 breast cancer cells in order to determine any
correlation between toxicity and DNA methylation and estrogen receptor binding. The
results of these studies are presented.
References
0
20
40
60
80
100
120
140
0 20 40 60 80 100 120
%Control(CellSurvival)
Concentration (uM)
Cell Survival
Melex
1a
2a
3a
Linear (Melex)
Linear (1a)
Linear (2a)
Linear (3a)
0
20
40
60
80
100
120
140
0 20 40 60 80 100 120
%Control(CellSurvival)
Concentration (uM)
Cell Survival - Control
Control
1b
2b
3b
Linear (Control)
Linear (1b)
Linear (2b)
Linear (3b)
Compound IC50 (nM) Relative Binding Affinity
Estradiol 0.58 100%
1b 0.99 58%
2b 1.48 39%
3b 1.11 52%
Synthesis of Molecules3
• Toxicity of compounds was determined in
MCF-7 breast cancer cells that
overexpress estrogen receptor-α.
• All methyl-sulfonates were toxic to MCF-
7 cells, with compound 2a being the most
toxic (EC50 ≈ 40 μM ), followed by 3a,
and then 1a.
• All sulfone analogues were not toxic to
MCF-7 cells.
• 3Me-A is the predominant adduct formed by all
three compounds
• Compound 2a produces significantly higher
levels of 3-MeA relative to the other
compounds.
Possible explanation for the variation in
DNA binding of compounds.
1. Franza, Gilberto, and Barry Gold. "The Biological Effects of N3-
methyladenine." Journal of Cellular Biochemistry 19.2 (2004): 250-257. Web. Aug. &
Sept. 2013.
2. Charles Kelly
3. Kishton, Rigel, Sean E. Miller, Heather Perry, Tera Lynch, Mayur Patel, Vinayak K. Gore,
and Giridhar R. Akkaraju. "DNA Site-specific N3-adenine Methylation Targeted to
Estrogen Receptor-positive Cells." Biorganic and Medicinal Chemistry 195093-5102.
This project was funded in part by: the NC Biotechnology Center Research Grant (#CC6425),
the Research Corporation (#2008BRG1214), the GlaxoSmithKline Summer Undergraduate
Research Scholarship, and the UNCW CSURF Research Supplies award, UNCW CSURF
Undergraduate Fellowships, and the UNCW Charles L. Cahill Award. We also thank Dr.
Suzanne Wardell and Dr. Donald McDeonnell for help with gene transcription studies.
Compound 2
Compound 1
Table 1
DNA adduct levels obtained upon reaction of compounds
with calf thymus DNAa
Comp-
ound
conc
µM
netrop
µM
adduct level
µmol adduct/mol DNA
3-MeA 7-MeG
1 25 1615 ± 45 194 ± 35
50 1764 ± 17 280 ± 36
100 1922 ± 35 404 ± 109
100 100 180 ± 10 482 ± 57
2 25 6523 ± 363 93 ± 31
50 11131 ± 30 253 ± 10
100 14449 ± 161 495 ± 35
100 100 769 ± 42 409 ± 3
3 25 699 ± 19 147 ± 20
50 922 ± 31 204 ± 25
100 1206 ± 23 318 ± 15
100 100 173 ± 16 420 ± 73
Me-lex 25 4599 ± 385 185 ± 59
50 8234 ± 542 399 ± 108
100 14280 ± 1250 774 ± 73
100 100 657 ± 12 714 ± 67
MMS 5000 1799 ± 39 18067 ± 76
5000 100 1239 ± 88 18935 ± 502
a DNA (1 mM) was reacted with methylating compounds, in the
presence or absence of netropsin, for 24 h at RT in 10 mM sodium
cacodylate buffer (pH 7.0) containing 10% DMSO.
0
2000
4000
6000
8000
10000
12000
14000
16000
1 2 3 Me-lex MMS
Adduct levels (μmol adduct/mol DNA)
3-MeA
7-MeG
DNA Binding:
Fluorescent Intercalator Displacement Assay
Estrogen Receptor Gene Transcription
A. Duplex DNA
B. Ethidium bromide intercalated DNA
exhibits fluorescence enhancement.
C. EtBris displaced by ligand and
fluorescence diminishes.
800000
1000000
1200000
1400000
1600000
1800000
2000000
1 2 3 4 5 6 7
FluorescenceIntensity
molar eq. compound
DNA Binding Assay
4.72 eq.
4.43 eq.
2.54 eq.
1b
2b
3b
• Compounds act as SERMS.
• Stereoview of compound 3a bound in the
minor groove of DNA2